TCR-seq

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Workflow

Calling Clonotypes

Calling method from contigs

1. Using Gene information

  • VDJC(TRA/TRB)
  • CDR3nt
  • CDR3aa
  • Combination

2. Using Chain information

  • TRA1+TRB1
  • TRA1 or TRB1
  • multi-chains

Basic Clonotype Analysis

Immune repertoire analysis

1. Diversity Index

  • Shannon Entropy
  • Simpson

2. Repertoire analysis.

  • TRAV/D/J/C distribution
  • TRBV/D/J/C distribution
  • CDR3nt length distribution
  • Clustering of clonotype repertoire

3. Expansion study

  • Clonotype expansion (hyper,high,medium,small,rare)
  • Top 100 expanded clonotypes with TRAV:TRBV info


Target Cell:Clonotype Analysis

Combination with scRNA-seq (if available)

1. Target cell selection

  • Cells with target features (e.g. cytotoxic cells, effector T cells, NKT, etc).
  • Cells associated with expanded clonotypes (hyper, high, medium, small, rare).
  • Cells with clonotypes displaying treatment-specific response.

2. Correlation of clonotype frequency with related data

  • Correlation of clonotype frequency with target gene expression profiles.
  • Correlation with clinical outcomes and phenotypic data. (if available)

3. Comparative Analysis

  • Comparison between different patient groups (e.g., responders vs non-responders to therapy, different disease stages) and associated clonotypes.
  • Clonotype repertoires to identify conserved features of the immune response.

4. Statistical and Computational Approaches

  • Application of statistical models and computational algorithms for the analysis.
  • Evaluation of the robustness and reproducibility of findings.



Example Plots

Clonotype Distribution



Clonotype Abundance


Sample A

Sample B


Cytotoxic Gene Expression Profiling across Clonotypes and Samples

Important Cytotoxic Gene Information

NKG7
- Natural Killer Growth Factor 7, the function of cytotoxic lymphocytes, particularly Natural Killer (NK) cells, potentially through the regulation of apoptotic pathways.

KLRG1
- Killer Cell Lectin-Like Receptor G1, is a transmembrane protein receptor primarily expressed on cytotoxic lymphocytes, regulating their activation and effector functions.

IFNG
- Interferon Gamma, is a cytokine crucially involved in the activation and effector functions of cytotoxic lymphocytes, including NK cells and cytotoxic T cells. IFNG enhances the cytotoxicity of these cells, promotes antigen presentation, and directs T helper cell differentiation towards a Th1 phenotype, crucial for effective immune responses.

GZMA
- Granzyme A, is a serine protease predominantly expressed in cytotoxic lymphocytes, facilitating target cell apoptosis.Upon recognition of target cells, GZMA is released and initiates caspase-independent cell death pathways, leading to the elimination of infected or transformed cells.

PRF1
- Perforin-1, is a pore-forming protein primarily expressed in cytotoxic lymphocytes, essential for target cell lysis. PRF1 forms pores in target cell membranes, enabling the delivery of pro-apoptotic molecules and promoting target cell death, crucial for immune surveillance and defense.



Heatmap